Development of Cryopreservation Technique for Vascularized Skin

Extensive soft-tissue defects and lack of donor tissue availability may require reconstruction with composite tissue allotransplantatiion (CTA). Currently, the main obstacles for CTA are immunogenicity, lack of a donor population, and organ storage limitations. Crypreservation of CTA could resolve these difficulties and broaden the application of CTA.

Twenty-two vascularized groin flap transplantations were performed between Lewis rats. Different cryoprotectants were used: DMSO in 18 flaps (Group 1) and glycerol in 4 flaps (Group 2). In 12 animals from Group 1, a streptokinase (STK) protocol was introduced which included perfusion with STK solution, 5000 IU before freezing and after thawing. Additional 1000 IU doses were administered systematically in the recipient rats after vessel anastomosis. Femoral arteries were cannulated and attached to a perfusion pump with the pressure limit set on 200 mmHG and flow rate at 80 ml/h. Cryoprotectant solutions were delivered via the femoral artery. Flaps were frozen up to − 196° C using a stepwise technique. After 3 days average, flaps were thawed. Group 1 flaps were perfused with sucrose solutions in decreasing concentrations. Group 2 flaps were perfused with Ringer's lactate. Flaps were transplanted as isografts to the groin region of the recipients and the femoral vessels were anastomosed in an end-to-end manner. Flap survival was evaluated by the patency of vascular anastomoses, flap skin color, and capillary refill. Biopsies of the flaps were taken for histologic evaluation.

During post thawing perfusion, flap edema was observed in all 18 DMSO cryopreserved flaps. In all cases where STK was not introduced (10 flaps), early arterial thrombosis occurred and capillary refill was not observed, with subsequent flap failure. Administration of streptokinase (12 flaps) extended arterial patency and allowed flap perfusion and capillary refill. In one flap, 2-week survival was achieved; in 9 flaps, necrosis occurred within 3 days post transplantation; 2 animals died within 16 hr post STK administration.

Cryopreservation in a vascularized groin flap model is a challenging task. Use of new cryoprotective solutions and fine-tuning of the thawing process may be keys to success and longer flap survival. An anticoagulation treatment was essential in prevention of early pedicle thrombosis. Further studies on alterations of perfusion, freezing and thawing protocols are in progress, in order to achieve dependable cryopreservation protocols for storage of vascularized skin allotransplants.